Process for the biological production of l-pipecolic acid

ABSTRACT

A process for the production of L-pipecolic acid which comprises the step of reducing delta-1-piperideine-6-carboxylic acid by the use of pyrroline-5-carboxylate reductase. A recombinant bacterium which can be used in this production process is also provided. Thus, the present invention can provide an efficient biological process for the production of L-pipecolic acid (or 2-piperidinecarboxylic acid).

TECHNICAL FIELD

[0001] This invention relates to a biological process for the productionof L-pipecolic acid (or 2-piperidinecarboxylic acid or L-homoproline)and to recombinant strains of Escherichia coli or coryneform bacteriawhich can conveniently be used for the process.

BACKGROUND ART

[0002] L-Pipecolic acid is important as a raw material for the synthesisof drugs. At present, L-pipecolic acid is being produced by synthesisfrom L-lysine (J. Chem. Soc. Chem. Commun., 1985, pp. 633-635) or by theoptical resolution of DL-pipecolic acid prepared by synthesis frompicolinic acid (Method of Enzymol., 17B, pp. 174-188, 1971). As methodsfor optical resolution, there are known a diastereomer salt method usingD-tartaric acid and an enzymatic method in which D-amino acid oxidasederived from pig liver is used to decompose the D-isomer while leavingthe L-isomer.

[0003] On the other hand, it is known that L-pipecolic acid is producedin animals (J. Biol. Chem., Vol. 211, p. 851, 1954), plants (J. Amer.Chem. Soc., Vol. 74, p. 2949, 1952) and microorganisms (Biochemistry,Vol. 1, pp. 606-612, 1926; Japanese Patent Laid-Open No. 38781/'94).However, since the amount of L-pipecolic acid accumulated therein issmall, no process for the production of L-pipecolic acid by using theseorganisms has been put to practical use. From previous investigations onthe metabolism of L-lysine, it is known thatdelta-1-piperideine-6-carboxylic acid (hereinafter also referred to asP6C) is formed from L-lysine through a transamination reaction by lysine6-aminotransferase (hereinafter also referred to as LAT) (Biochemistry,Vol. 7, pp. 4102-4109, 1968) or by the action of L-lysine6-dehydrogenase (J. Biochem., Vol. 105, pp. 1002-1008, 1989).

[0004] It has been reported P6C can be chemically converted intoL-pipecolic acid by hydrogenation using platinum oxide (Biochemistry,Vol. 7, pp. 4102-4109, 1968), but there is no report about the formationof L-pipecolic acid by the biological or enzymatic reduction of P6C.Moreover, a metabolic pathway is supposed in which Pseudomonas putidaproduces L-pipecolic acid from D-lysine viadelta-1-piperideine-2-carboxylic acid. It is also difficult to utilizesuch biological pathways for the mass production of L-pipecolic acid.

[0005] In the above-described process involving the optical resolutionof DL-pipecolic acid prepared by chemical synthesis, the opticalresolving agent used is expensive and a complicated procedure isrequired. Moreover, in the process using an enzyme for purposes ofoptical resolution, the use of a purified enzyme is also expensive.Because of these disadvantages, both processes are not efficient from anindustrial point of view and cannot produce L-pipecolic acid cheaply.

[0006] Furthermore, conventional processes for the production ofL-pipecolic acid by using microorganisms have not been put to practicaluse because the amount of L-pipecolic acid accumulated is small.

DISCLOSURE OF THE INVENTION

[0007] The present inventors have now found that pyrroline-5-carboxylatereductase [EC 1.5.1.2], which reduces delta-1-pyrroline-5-carboxylicacid to L-proline as shown below,

[0008] can also reduce P6C to the corresponding L-pipecolic acidefficiently as shown below.

[0009] Moreover, it has also been found that this reduction system maybe used by combining it conveniently with other biological P6Cproduction systems.

[0010] The present invention is based on these findings and provides ameans for producing L-pipecolic acid efficiently by utilizing the actionof pyrroline-5-carboxylate reductase.

[0011] Accordingly, the present invention relates to a process for theproduction of L-pipecolic acid which comprises the step of reducingdelta-1-piperideine-6-carboxylic acid (P6C) by the use ofpyrroline-5-carboxylate reductase.

[0012] In a preferred embodiment of the present invention, the P6Creduction step is combined with the step of converting L-lysine into P6Cby the use of lysine 6-aminotransferase (LAT).

[0013] The present invention also relates to a recombinant strain ofEscherichia coli or a coryneform bacterium which contains a geneencoding LAT in expressible form.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a schematic view of a plasmid related to the presentinvention which has been constructed in order to produce L-pipecolicacid from L-lysine in Escherichia coli

[0015]FIG. 2 is a schematic view of a plasmid related to the presentinvention which has been constructed in order to produce L-pipecolicacid in a coryneform bacterium.

BEST MODE FOR CARRYING OUT THE INVENTION

[0016] The term “exogenous gene” as used herein means a gene derivedfrom a cell different from the mentioned cell itself, whether the cellis similar or dissimilar to the mentioned cell or bacterium.

[0017] Pyrroline-5-carboxylate reductase (EC 1.5.1.2; hereinafter alsoreferred to as P5C reductase), which is used in the present invention,is commonly known to be an enzyme which participates in a metabolicpathway for synthesizing proline from arginine or glutamic acid. Asdescribed above, this enzyme has an activity for reducingdelta-1-pyrroline-5-carboxylic acid to proline with the aid of thereduced form of nicotinamide adenine dinucleotide (NADH) or the reducedform of nicotinamide adenine dinucleotide phosphate (NADPH). It is knownthat P5C reductase is widely distributed in a great variety of bacteria,plants and animals.

[0018] P5C reductase that can be used in the present invention is notlimited by its origin, so long as it has an activity for reducing P6C toL-pipecolic acid. P5C reductase may be used in any desired form selectedfrom preparations such as a purified enzyme and a cell lysate, and fromliving cells. When a preparation is used, the addition of NADH or NADPHmay sometimes be needed to carry out the reduction in accordance withthe present invention.

[0019] Delta-1-piperideine-6-carboxylic acid (P6C), which is to bereduced by the use of P5C reductase according to the present invention,corresponds to a compound obtained when 2-aminoadipic acid6-semialdehyde formed from L-lysine by the action of lysine6-aminotransferase (LAT) undergoes nonenzymatic ring closing with theelimination of water. Since this semialdehyde is usually considered tobe present in an aqueous solution as an equilibrium mixture with P6C, itis understood that P6C and the semialdehyde are equivalent to each otherin the reaction system of the present invention. Accordingly, P6Citself, a mixture of P6C and the semialdehyde, or the semialdehydeitself may be added to the reaction system of the present invention, andall of these embodiments are comprehended in the present invention.

[0020] As P6C (or 2-aminoadipic acid 6-semialdehyde), there may be usedany of the products prepared by various means including chemicalsynthesis and biological means. However, from the viewpoint of theeconomical production of L-pipecolic acid that is a particular opticalisomer, it is preferable to use P6C having a steric configurationcorresponding to that of L-pipecolic acid (specifically, with respect tothe asymmetric carbon atom located at the 2-position).

[0021] The step of reducing P6C according to the present invention iscarried out by making P5C reductase act on P6C under conditions whichallow ordinary enzyme reactions to proceed. As described above, P5Creductase may be made to act on P6C in any desired form selected frompreparations such as a purified enzyme and a cell lysate, and fromliving cells. However, in view of the fact that a coenzyme such as NADHor NADPH participates in the aforesaid reaction, it is preferable to usea cell lysate or living cells themselves. As the cells, it is especiallypreferable to use cells of Escherichia coli or a coryneform bacterium,among microorganisms exhibiting P5C reductase activity capable ofconverting (or reducing) P6C into L-pipecolic acid. It is known that theproC gene encoding P5C reductase is present in Escherichia coli, and thesequence of proC and its expression have been reported (see A. H. Deutchet al., Nucleic Acids Research, Vol. 10, 1982, 7701-7714).

[0022] Accordingly, in a preferred embodiment of the present invention,P6C can be reduced to L-pipecolic acid by incubating Escherichia colihaving P5C reductase activity, or Escherichia coli or anothermicroorganism containing the aforesaid proC gene in expressible form,together with P6C under conditions which allow these microorganisms tolive and produce P5C reductase activity. As used herein, the expression“containing or integrating a particular gene in expressible form” meansthat the gene is integrated into a chromosome of a host cell, ifnecessary, together with a promoter, a regulator and the like; or thatthe gene is integrated into an expression vector together with asuitable promoter and the like, and then introduced into a host cell.The aforesaid conditions which allow the microorganisms to produce P5Creductase activity refer to conditions under which the respectivemicroorganisms are viable and preferably culture conditions under whichthey can grow. These conditions are well known to those skilled in theart, and it would be easy for the those skilled in the art to determinedthe conditions with reference to the examples which will be given later.

[0023] The desired reaction may be carried out by adding P6C directly toa suspension or culture of a microorganism as described above. However,according to the present invention, it is preferable to feed P6C to theaforesaid reduction step using P5C reductase by combining it with thestep of converting readily available L-lysine into P6C by the use oflysine 6-aminotransferase (LAT). Where Escherichia coli is used as asource of P5C reductase in such a combination, it is usually necessaryto use a LAT enzyme system derived from foreign cells in combinationwith the enzyme system of Escherichia coli, because an enzyme systemcatalyzing the process of converting L-lysine into P6C is not present inEscherichia coli or, even if it is present, its activity is very low.LAT that can be used in such a combination is not limited by its origin,provided that it has an activity for converting L-lysine into P6C. Apreferred example is LAT derived from Flavobacterium lutescens. Typicalstrains (e.g, IFO 3084 strain) of this microorganism are usually usedfor the bioassay of L-lysine and are known to have LAT activity [Soda etal., Biochemistry, 7(1968), 4102-4109; Ibid., 4110-4119]. Certainstrains of F. lutescens have the ability to oxidize P6C to α-aminoadipicacid by the action of delta-1-piperideine-6-carboxylate dehydrogenasepossessed thereby [Biochem. J. (1977), 327, 59-64]. Moreover, there is apossibility that L-pipecolic acid formed from P6C by P5C reductase orP6C reductase activity may be converted into other compounds through afurther metabolic pathway. Consequently, when the enzyme system of thisbacterium is used, it may usually happen that the conversion of L-lysineinto L-pipecolic acid cannot be achieved or, even if this conversionoccurs, L-pipecolic acid is not accumulated. Accordingly, in order toaccomplish the purpose of the present invention, it is preferable to usea combination of enzyme systems derived from different types of cells(or microorganisms) as described above. Microorganisms having such acombination of enzyme systems include, but are not limited to, theaforesaid Escherichia coli into which, for example, the lat gene of F.lutescens encoding LAT is introduced in expressible form; conversely, F.lutescens into which the proC gene of Escherichia coli is introduced;and host microorganisms which can be suitably used for other purpose ofthe present invention and in which both lat and proC are introduced inexpressible form. Each gene may be introduced into the host by means ofa recombinant plasmid, or may be integrated directly into a chromosomeof the host in expressible form. When F. lutescens is used as the host,there is a possibility that this microorganism may metabolize the formedL-pipecolic acid through a further metabolic pathway. Consequently, itmay be necessary to use a variant in which such a metabolic pathway isblocked. As the enzyme system suitable for the purpose of convertingL-lysine into L-pipecolic acid according to the present invention, anenzyme system constructed by using Escherichia coli (also serving as asource of proC in some cases) as the host and introducing thereinto atleast the lat of F. lutescens can conveniently be used owing to thestability of the system, its ease of treatment, its high conversionefficiency and the like. Furthermore, when Escherichia coli is used asthe host, an exogenous proC gene may be introduced in addition to thelat gene. Especially for the purpose of facilitating the incorporationof the starting material (i.e., L-lysine) into bacterial cells, it ispreferable to introduce an exogenous gene encoding the lysine-specificincorporation (or permeation) enzyme of Escherichia coli (also referredto as a gene participating in the incorporation of lysine). Typicalexamples of such a gene include, but are not limited to, the gene (lysP)encoding a lysine-specific permease, and the genes (argT, hisP and hisQ)encoding proteins constituting the LAO system participating in theincorporation of lysine, arginine and ornithine. For example, accordingto J. Bacteriol., Vol. 174, 3242-3249, 1992, it is suggested thatEscherichia coli into which the lysP gene has been introduced by meansof a multicopy plasmid shows a 20-fold increase in lysine incorporationrate.

[0024] The above-described systems, which comprise combinations ofenzyme systems or genes and can be used in the present invention, may beconstructed or prepared according to techniques which are related tocytobiology, cell culture, molecular biology, microbiology andrecombinant DNAs and are commonly used per se by those skilled in theart. As to these techniques, reference may be made, for example, toMolecular Cloning—A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al., U.S. Pat. No. 4,683,195; and NucleicAcid Hybridization (B. D. Hames & S. J. Higgins eds., 1984).

[0025] The construction or preparation of enzyme systems ormicroorganisms which can preferably be used in the present invention ismore specifically described below in connection with embodiments inwhich Escherichia coli (hereinafter abbreviated as E. coli). However, itis to be understood that the present invention is not limited by theseembodiments.

[0026] Host-Vector System:

[0027] Commercially available strains and vectors may be suitably used.The embodiments described herein are such that Escherichia coliBL21(DE3), Escherichia coli BL21 or Escherichia coli C600 strain is usedas the host and a pET or pUC system is used as the vector. As othervectors, the vectors conforming to “Guidelines for the Industrializationof Recombinant DNA Technology” (the Ministry of International Trade andIndustry), Guideline, Section 2, No. 3, 2.(2) may preferably be used.

[0028] Cloning and Expression of the lat Gene:

[0029] LAT was purified from the culture supernatant of F. lutescens IFO3084 strain by hydrophobicity chromatography, ion exchangechromatography and gel permeation chromatography. The enzyme activity ofLAT was measured by a colorimetric method using o-aminobenzaldehyde(Biochemistry, Vol. 7, pp. 4102-4109, 1968). The N-terminus of thepurified LAT was determined to be KLLAPLAPLRAHAGTRLTQGL. On the basis ofthis amino acid sequence, mix primers were designed and used in PCR toamplify DNA fragments of lat from the genomic DNA of F. lutescens IFO3084 strain. Then, on the basis of the DNA fragments thus obtained, theentire lat gene of about 1.6 kbp size was obtained by inverse PCR. TheDNA sequence of lat and the amino acid sequence of LAT are shown in SEQID NO: 1. For further details about the cloning of the lat gene,reference may be made to International Application PCT/J99/04197 that iscopending with the present application, if necessary.

[0030] From the so-determined base sequence of the lat gene, thefollowing forward DNA primer in which a region in the neighborhood ofthe N-terminal ATG of the lat gene was altered to a NdeI site ETlaNdeF:TCCATATGTCCCTTCTTGCCCCGCTCGCCC (SEQ ID NO:2)

[0031] and the following reverse DNA primer in which a region downstreamof the termination codon thereof was altered to a BamHI site

[0032] ETlaBamR: GCGGATCCTGTTGCCGCTGGTGCCGGGCAG (SEQ ID NO: 3)

[0033] were prepared. Using these primers, PCR was carried out toamplify the lat gene region of about 1.6 kbp size. This amplifiedfragment was digested with the restriction enzymes NdeI and BamHI toprepare an insert DNA solution. On the other hand, the expression vectorpET11a (manufactured by Novagen) was digested with the restrictionenzymes NdeI and BamHI, and subjected to a ligation reaction with theinsert DNA solution by means of Ligation Kit version 2 (manufactured byTaKaRa). The plasmid thus obtained was named pETlat. pETlat is a plasmiddesigned so as to bring about the expression of natural LAT protein. E.coli BL21(DE3) was transformed with this plasmid, and the resultingstrain was named E. coli BL21(DE3)pETlat strain.

[0034] Next, the expression vector pET11a was replaced by pUC19, and E.coli BL21 strain was used as the host. From the aforesaid base sequenceof the lat gene, the following forward DNA primer in which a region inthe neighborhood of the N-terminal ATG of the lat gene was altered to aHindIII site lathiF19: ATAAGCTTGTCCCTTCTTGCCCCGCTCGC (SEQ ID NO:4)

[0035] was prepared and the following reverse DNA primer in which aregion downstream of the termination codon thereof was altered to aBamHI site ETlaBamR: GCGGATCCTGTTGCCGCTGGTGCCGGGCAG (SEQ ID NO:3)

[0036] were prepared. Using these primers, PCR was carried out toamplify the lat gene region of about 1.6 kbp size. This amplifiedfragment was digested with the restriction enzymes HindIII and BamHI toprepare an insert DNA solution. On the other hand, the vector pUC19 wasdigested with the restriction enzymes HindIII and BamHI, and subjectedto a ligation reaction with the insert DNA solution by means of LigationKit version 2 (manufactured by TaKaRa). The plasmid thus obtained wasnamed pUClat (see FIG. 1). pUClat is a plasmid designed so as to bringabout the expression of LacZ-LAT fusion protein. E. coli BL21 wastransformed with this plasmid, and the resulting strain was named E.coli BL21pUClat strain.

[0037] When each of E. coli BL21(DE3)pETlat strain and E. coliBL21pUClat strain was cultured in a culture medium (1.5% Bacto tryptone,3.0% yeast extract, 0.5% glycerol, pH 7) containing L-lysine, theaccumulation of L-pipecolic acid in the culture medium was observed inboth cases. This means that an enzyme capable of reducing P6C formedfrom L-lysine by a transamination reaction catalyzed by LAT is presentin Escherichia coli used as the host.

[0038] A search was made for this P6C reduction enzyme. From the geneticinformation on the whole genome of Escherichia coli, it was supposedthat the P5C reduction enzyme also reduced P6C. Then, the role of proCin L-pipecolic acid production was investigated by using E. coli RK4904strain (obtained from the E. coil Genetic Stock Center of YaleUniversity) that is a proC-deficient proC32 mutant strain. First, inorder to examine the effect of proC (see SEQ ID NO: 5), it was tried tointroduce proC into pUClat. The following DNA primers having a KpnI siteattached to an end thereof were prepared. procKpnF:AGGGTACCATAAAATCGCGCATCGTCAGGC (SEQ ID NO:6) procKpnR:CCGGTACCGCCACAGGTAACTTTACGGATT (SEQ ID NO:7)

[0039] Using these primers, PCR was carried out to amplify aproC-containing fragment of about 1.5 Kbp size. This amplified fragmentof about 1.5 Kbp size was digested with the restriction enzyme KpnI toprepare an insert DNA solution. On the other hand, the plasmid pUClatwas digested with the restriction enzyme KpnI, and subjected to aligation reaction with the insert DNA solution by means of Ligation Kitversion 2 (manufactured by TaKaRa). The resulting plasmid in which latand proC are ligated so as to be oriented in the forward direction wasnamed pUClatproC.

[0040]E. coli RK4904 was transformed with this plasmid, and theresulting strain was named E. coli RK4904pUClatproC strain. Moreover, aplasmid having proC alone was prepared. Specifically, pUClatproC wasdigested with the restriction enzymes BamHI and HindIII, blunt-ended bymeans of Blunting Kit (manufactured by TaKaRa), and subjected to aself-ligation reaction. The plasmid thus obtained was named pUCproC. E.coli RK4904 was transformed with this plasmid, and the resulting strainwas named E. coli RK4904pUCproC strain. When L-pipecolic acid productiontests were carried out by using the so-constructed E. coli RK4904pUC19strain, E. coli RK4904pUClat strain, E. coli RK4904pUCproC strain and E.coli RK4904pUClatproC strain, E. coli RK4904pUClatproC strain aloneshowed the accumulation of L-pipecolic acid and the other strains showedno production of L-pipecolic acid.

[0041] These results indicate that L-pipecolic acid is produced onlywhen both lat and proC are expressed in E. coli and that P5C reductase,which is a protein encoded by proC, also reduces P6C. To the presentinventors' knowledge, no enzyme capable of reducing P6C has beendescribed in the literature, and the present description discloses suchan enzyme for the first time.

[0042] Cloning of the lysP Gene and Cointegration of the lysP and latGenes:

[0043] According to the aforementioned J. Bacteriol., Vol. 174,3242-3249, 1992, there is a possibility that the rate of theincorporation of lysine into Escherichia coli determines the rate of theproduction of L-pipecolic acid by Escherichia col. Now, as describedbelow, it was tried to introduce the lysP gene encoding alysine-specific permease into the plasmid pETlat. From the geneticinformation on the sequence of the lysP gene of Escherichia coli (seeSEQ ID NO: 8), the following DNA primers having Bg/II and BamHI sitesattached to an end thereof were prepared. lysPBgBmF:TGAGATCTGGATCCTGCGTGAACGCGGTTC (SEQ ID NO:9) lysPBgBmR:GCAGATCTGGATCCCAGAAAGCCGGAACAG (SEQ ID NO:10)

[0044] For the cloning of lysP, PCR was carried out by using theseprimers to amplify a lysP containing fragment of about 2.2 Kbp size.This amplified fragment of about 2.2 Kbp size was digested with therestriction enzyme Bg/II to prepare an insert DNA solution. On the otherhand, pETlat was digested with the restriction enzyme Bg/II, andsubjected to a ligation reaction with the insert DNA solution by meansof Ligation Kit version 2 (manufactured by TaKaRa). The so-constructedplasmid in which lat and lysP are ligated so as to be oriented in theopposite directions was named pETlatlysP. E. coli BL21(DE3) wastransformed with this plasmid, and the resulting strain was named E.coli BL21(DE3)pETlatlysP strain. When L-pipecolic acid production testswere carried out by using this E. coli BL21(DE3)pETlatlysP strain and E.coli BL21(DE3)pETlat strain (obtained by transformation with thepreviously prepared pETlat), it was confirmed that E. coliBL21(DE3)pETlatlysP strain produced L-pipecolic acid in a three timesgreater amount than E. coli BL21(DE3)pETlat strain.

[0045] Moreover, the aforesaid amplified fragment of about 2.2 Kbp sizewas digested with the restriction enzyme BamHI to prepare an insert DNAsolution. On the other hand, pUClat was digested with the restrictionenzyme BamHI, and subjected to a ligation reaction with the insert DNAsolution by means of Ligation Kit version 2 (manufactured by TaKaRa).The so-constructed plasmid in which lat and lysP are ligated so as to beoriented in the forward direction was named pUClatlysP (see FIG. 1). E.coli BL21 was transformed with this plasmid, and the resulting strainwas named E. coli BL21pUClatlysP strain. When L-pipecolic acidproduction tests were carried out by using this E. coli BL21pUClatlysPstrain and E. coli BL21pUClat strain, it was confirmed that E. coliBL21pUClatlysP strain produced L-pipecolic acid in a three times greateramount than E. coli BL21pUClat strain. Thus, when Escherichia coli isused as the host, it is desirable to introduce the lysP gene thereinto.E. coli BL21pUClatlysP strain in accordance with the present inventionwas deposited on Dec. 20, 1999 with the National Institute of Bioscienceand Human-Technology, the Agency of Industrial Science and Technology(1-3, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan) andassigned the accession number FERM P-17681. Thereafter, the aforesaiddeposition was transferred to the international deposition department ofthe institute under the provisions of the so-called Budapest Treaty andassigned the accession number FERM BP-7326.

[0046] Introduction of the yeiE Gene:

[0047] In order to improve the L-pipecolic acid-producing ability ofBL21pUClatlysP strain, it was tried to further enhance the activity oflysP. The E. coli genome project has revealed the DNA sequence of aregion around lysP This indicates that the yeiE gene (see SEQ ID NO: 11)arranged in tandem with lysP is present on the upstream side of lysP.From its amino acid sequence, it is suggested that yeiE is a lysR typetranscriptional regulator sequence which is frequently retained inbacteria. Since it was supposed that this yeiE might control thetranscription of lysP, it was expected that the L-pipecolicacid-producing ability could be improved by integrating both yeiE andlysP into a plasmid to increase the transcription of lysP and therebyenhance the ability to incorporate L-lysine.

[0048] The plasmid pUClatlysPL was constructed as described below. Thefollowing forward DNA primer having a Bg/II site attached to an endthereof ATAGATCTCTTGTTGCCTAAAACCATCCCCAA (SEQ ID NO:12)

[0049] and the following reverse DNA primer having a KpnI site attachedto an end thereof GTGGTACCCCCCAGAAAGCCGGAACAGCCTC (SEQ ID NO:13)

[0050] were prepared. Using these primers, PCR was carried out toamplify a yeiE- and lysP-containing region of about 3 Kbp size. Thisamplified fragment was digested with the restriction enzymes Bg/III andKpnI to prepare an insert DNA solution. On the other hand, pUClatlysPwas digested with the restriction enzymes BamHI and KpnI, and subjectedto a ligation reaction with the insert DNA solution by means of LigationKit version 2 (manufactured by TaKaRa). The plasmid thus obtained wasnamed pUClatlysPL (see FIG. 1). E. coli BL21 was transformed with thisplasmid, and the resulting strain was named E. coli BL21pUClatlysPLstrain.

[0051] Cloning and Introduction of the argT Gene:

[0052] It is known that the expression of lysP is suppressed at highlysine concentrations and induced at low lysine concentrations [J.Bacteriol. (1996), Vol., 178, 5522-5528]. Accordingly, it was planned tointroduce an additional gene participating the incorporation of lysineinto cells. Up to this time, it is known that a system for theincorporation of lysine, arginine and ornithine into cells (i.e., theLAO system) is present in Escherichia coli [Journal of BiologicalChemistry, Vol. 265, pp. 1783-1786 (1990)]. Moreover, since the argTgene of Escherichia coli clarified by the genome project has highhomology with the argT gene of Salmonella typhimurium shown toparticipate in the LAO system [Proc. Natl. Acad. Sci. USA, Vol. 78, pp.6038-6042 (1981)], it was expected that the argT gene of Escherichiacoli was highly likely to participate in the incorporation of lysine.

[0053] Now, in order to examine the effect of argT, the plasmidpUClatargT having lat and argT integrated thereinto were constructed asdescribed below. The following primers were prepared. (SEQ ID NO:14)argTkpnF: TCGGTACCTCGACATTTTGTTTCTGCC (SEQ ID NO:15) argTkpnR:ATGGTACCATAAAATTGACCATCAAGG

[0054] Using these primers and a template comprising the genomic DNA ofEscherichia coli, PCR was carried out to amplify argT. The reactionconditions were such that one cycle consisted of 98° C./20 seconds, 60°C./30 seconds and 68° C./1 minute, and this cycle was repeated 25 times.This amplified fragment of about 1.5 Kbp was digested with therestriction enzyme KpnI and inserted into the KpnI site of pUClatlysPL.Moreover, the tetracycline resistance gene was inserted into the ScaIsite of the resulting plasmid. Specifically, the following primershaving a ScaI site added to an end thereof were prepared. TetF:TTAGTACTCTTATCATCGATAAGCTTTAAT (SEQ ID NO:16) TetR:GCAGTACTACAGTTCTCCGCAAGAATTGAT (SEQ ID NO:17)

[0055] Using these primers and a template comprising pBR322, PCR wascarried out to amplify the tetracycline resistance gene. This gene wasinserted into the ScaI site present in the ampicillin resistance gene ofpUClatlysPL, and the resulting plasmid was named pUClatlysPLargT-tet.This plasmid was introduced into E. coli BL21 strain, and the resultingstrain was named E. coli BL21pUClatlysPLargT-tet strain. The DNAsequence and amino acid sequence of argT are shown in SEQ ID NO: 18.

[0056] Construction and Expression of a lat Transformant in a CoryneformBacterium:

[0057] As described above, there has been established an L-pipecolicacid production system based on the conversion of L-lysine intoL-pipecolic acid by the use of a lat-expressing strain of Escherichiacoli. In this system using a recombinant strain of Escherichia coli, ithas been suggested that, in some cases, the incorporation of L-lysineinto cells determines the rate of production of L-pipecolic acid.Accordingly, it may be desirable to provide a direct L-pipecolicacid-producing bacterium which produces L-lysine by itself and convertsit into pipecolic acid without requiring the addition of L-lysine to theculture medium. This provision can be accomplished according to thefollowing strategy. L-lysine is produced in large amounts byfermentation with Corynebacterium glutamicum. Then, if lat can beintegrated into the pC2 plasmid [PLASMID, Vol. 36, 62-66 (1996)]established as a vector system for C. glutamicum and this plasmid can beintroduced into C. glutamicum ATCC31831 strain to bring about theexpression of lat, L-lysine biosynthesized in its cells will beconverted into P6C and this P6C will further be converted into pipecolicacid by the action of pyrroline-5-carboxylate reductase produced by theexpression of proC encoded in the genome of C. glutamicum. The validityof this strategy can be ascertained, for example, by carrying out thefollowing experiment.

[0058] Using the aforesaid plasmid pC2 plasmid, the plasmid pClat forthe expression of lat was constructed as described below. The followingforward DNA primers were prepared. (SEQ ID NO:19) ClatBamF:GGGGTACCCATGTCCCTTCTTGCCCCGCT (SEQ ID NO:20) ClatBamR:GGGGATCCCGCGGCCTGTTGCCGCTGGT

[0059] Using these primers and a template comprising the genomic DNA ofFlavobacterium lutescens, PCR was carried out to amplify lat. Thereaction conditions were such that one cycle consisted of 98° C./20seconds and 68° C./2 minutes, and this cycle was repeated 25 times. Thisamplified fragment of about 1.5 Kbp was digested with the restrictionenzymes KpnI and BamHI, and ligated into the KpnI and BamHI sites of pC2(FIG. 2). This plasmid pClat was introduced into E. coli JM109 strain.Using the strain thus obtained, it was confirmed that the conversion ofL-lysine into pipecolic acid proceeded, i.e. the strain had LATactivity.

[0060] Subsequently, C. glutamicum was transformed with pClat. Forexample, C. glutamicium was inoculated into 3 ml of L medium andincubated at 32° C. for 17 hours with shaking. 30 μl of the resultingculture was inoculated into 3 ml of L medium and incubated at 32° C. for2.5 hours with shaking. Then, 1.5 μl of a penicillin G potassiumsolution (2 mg/ml) was added thereto, and the incubation was continuedfor an additional 1.5 hours with shaking. The total amount of cells werecollected, washed with 5 ml of a 10% glycerol solution, and suspended in700 μl of a 10% glycerol solution to prepare an electro-cell suspension.0.2 μl of the plasmid dissolved in a TE solution (200 μg/ml) was addedto 200 μl of the electro-cell suspension, and subjected toelectroporation under conditions including 12.5 kV/cm, 25 μF and 200 Ω.After 1 ml of L medium was added thereto and this mixture was incubatedat 32° C. for 2 hours, the resulting culture was spread over an L platecontaining 10 μl/ml of kanamycin, and incubated at 32° C. for 3 days.Desired transformants can be obtained by screening the colonies soformed.

[0061] As used herein, the term “coryneform bacterium” comprehends anyspecies that typically belongs to the genus Corynebacterium, producesL-lysine, and meets the purposes of the present invention.

[0062] Alterations of the Above-Described Genes or DNA Sequences:

[0063] According to the present invention, the genes or DNAs encodingthe above-described various enzymes, i.e. the gene encoding lysine6-aminotransferase, the gene encoding pyrroline-5-carboxylate reductase,the gene encoding a lysine-specific incorporation enzyme, and the generegulating the transcription of lysP, also comprehend any alterationsthereof, provided that such alterations can hybridize with therespective genes (see, for example, SEQ ID NO: 1, SEQ ID NO: 5, SEQ IDNO: 8, SEQ ID NO: 11 and SEQ ID NO: 18) under stringent conditions andthe polypeptides produced by the expression thereof have respectivedesired enzyme activities.

[0064] Stringent conditions are well known to those skilled in the artand are described, for example, in the aforementioned manual by Sambrooket al., pp. 9.31-9.62. Such alterations may be made according to per seknown techniques such as point mutagenesis, site-directed mutagenesisand chemical synthesis, by examining, as an index, whether they have thedeletion or addition of one or more amino acids which are not essentialto the desired enzyme activity or exert no adverse influence thereon, orwhether they have a substitution between amino acid residues having asimilar side chain, such as amino acid residues having a basic sidechain (lysine, arginine, histidine, etc.), an acidic side chain(aspartic acid, glutamic acid, etc.), an uncharged polar side chain(glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,etc.), a nonpolar side chain (alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan, etc.), a β-branched sidechain (e.g., threonine, valine, isoleucine, etc.) or an aromatic sidechain (e.g., tyrosine, phenylalanine, tryptophan, histidine, etc.).Moreover, the transformation of host cells with such genes may becarried out according to the previously described procedures or methodswhich are well known to those skilled in the art.

[0065] In order to confirm that the resulting pipecolic acid was theL-form, HPLC using a chiral column (Agric. Biol. Chem., Vol. 52, pp.1113-1116, 1988) was employed. Using a DAICEL CHIRAL PAK WH (4.6×250 mm;manufactured by Daicel) as the column and a 0.25 mM aqueous solution ofcopper sulfate as the mobile phase, HPLC was carried out at a columntemperature of 50° C. and a flow rate of 1.0 ml/min. Detection wascarried out at an ultraviolet wavelength of 243 nm. Under theseconditions for HPLC, the retention time of D-pipecolic acid is 11.5minutes and the retention time of L-pipecolic acid is 15 minutes. Whenthe pipecolic acid produced by the recombinant strains of Escherichiacoli in accordance with the present invention was analyzed, theretention time of the pipecolic acid so produced was 15 minutes.

EXAMPLES

[0066] The present invention is more specifically explained withreference to the following examples.

[0067] In these examples, L-pipecolic acid was determined by dansylatingit and then subjecting it to high-performance liquid chromatography(hereinafter abbreviated as HPLC) while using proline as an internalstandard. Specifically, after the culture supernatant was diluted100-fold with distilled water, 10 μl was transferred to an Eppen tube,and 200 μl of a 40 mM lithium carbonate buffer (pH 9.5) containing 5μg/ml proline and 100 μl of an acetonitrile solution containing 3.0mg/ml dansyl chloride were added thereto. The resulting mixture wasstirred and reacted in the dark at room temperature for 2 hours. After10 μl of 2% methylamine hydrochloride was added thereto with stirring,the resulting supernatant was used as an analytical sample. As to theanalytical conditions for HPLC, the column was YMC-Pack ODS-A A-303(4.6×250 mm; manufactured by YMC), the mobile phase was a 33.2%acetonitrile solution containing 0.003 M L-proline, 0.0015 M coppersulfate and 0.0039 M ammonium acetate (adjusted to pH 7 with aqueousammonia), elution was carried out at a flow rate of 0.8 ml/min and atroom temperature, and detection was carried out at an excitationwavelength of 366 nm and a fluorescence wavelength of 510 nm. Underthese conditions, the retention time of L-pipecolic acid was 13 minutes.

Example 1

[0068] L-Pipecolic acid production tests were carried out with respectto E. coli BL21pUClatlysP (FERM BP-7326) strain, E. coliBL21(DE3)pETlatlysP strain, E. coli C600pUClatlysP strain and E. coliBL21pUClatlysPL strain. Each strain was inoculated into 3 ml of L medium(1.0% polypeptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, pH 7.2)containing 50 μg/ml ampicillin sodium, and incubated at 32° C. overnightwith shaking. The resulting cultures were used seed cultures, and 275 μlof each seed culture was inoculated into 27.5 ml of TB medium (0.44%glycerol, 1.33% Bacto-trypton, 2.67% Bacto-yeast extract, 0.21% KH₂PO₄,1.14% K₂HPO₄) containing 100 μg/ml ampicillin sodium, and incubated at32° C. for 4.5 hours with shaking. In the culture of a microorganismformed with a pET vector, such as E. coli BL21(DE3)pETlatlysP, 275 μl of100 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for thepurpose of inducing lat, and the incubation was continued at 32° C. foran additional 4 hours with shaking. In contrast, when a pUC vector wasused, the addition of IPTG was unnecessary. Subsequently, 500 μl of 50%L-lysine hydrochloride and 250 μl of 50% glycerol dissolved in aphosphate buffer (pH 6.8) were added to each culture, and the incubationwas continued at 32° C. with shaking. Furthermore, 15 hours, 39 hours,63 hours, 87 hours, 120 hours and 159 hours after the addition ofL-lysine hydrochloride, 500 μl of 50% L-lysine hydrochloride and 500 μlof 50% glycerol dissolved in a phosphate buffer (pH 6.8) were added. Onehundred-μl samples were taken after 15 hours, 39 hours, 63 hours, 87hours, 120 hours, 159 hours and 207 hours, and their pipecolic acidcontent was determined by HPLC. As a result, each strain accumulatedL-pipecolic acid in the culture medium at the following concentration.E. coli BL21pUClatlysP 10 g/l, E. coli BL21(DE3)pETlatlysP 26 g/l, E.coli C600pUClatlysP 0.8 g/l, and E. coli BL21pUClatlysPL 15 g/l.

Example 2

[0069] In order to elucidate the role of P5C reductase in L-pipecolicacid production, the ability to produce L-pipecolic acid was examinedwith respect to four recombinant strains formed by using proC-deficientE. coil RK4904 strain as the host, namely E. coli RK4904pUC19 strain, E.coli RK4904pUClat strain, E. coli RK4904pUCproC strain and E. coliRK4904pUClatproC strain. Each strain was inoculated into 3 ml of Lmedium (1.0% polypeptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose,pH 7.2) containing 50 μg/ml ampicillin sodium, and incubated at 32° C.overnight with shaking. The resulting cultures were used seed cultures,and 275 μl of each seed culture was inoculated into 27.5 ml of TB medium(0.44% glycerol, 1.33% Bacto-trypton, 2.67% Bacto-yeast extract, 0.21%KH₂PO₄, 1.14% K2HPO₄) containing 50 μg/ml ampicillin sodium, andincubated at 32° C. for 8 hours with shaking. Then, 500 μl of 50%L-lysine hydrochloride and 500 μl of 50% glycerol dissolved in aphosphate buffer (pH 6.8) were added thereto, and the incubation wascontinued at 32° C. with shaking. A 100-μl sample was taken after 40hours, and L-pipecolic acid was determined by HPLC. As a result, E. coliRK4904pUClatproC strain alone showed the accumulation of 0.765 g/l ofL-pipecolic acid, and the other strains showed no accumulation ofpipecolic acid.

Example 3

[0070] An investigation on the carbon source of the culture medium wascarried out by using E. coli C600pUClatlysP strain. As to thecomposition of the culture medium, there were used culture mediaobtained by substituting various carbon sources for the glycerol of TBmedium (0.44% glycerol, 1.33% Bacto-trypton, 2.67% Bacto-yeast extract,0.21% KH₂PO₄, 1.14% K₂HPO₄). As carbon sources, glycerol, sodiumpyruvate, citric acid, propionic acid, maleic acid, lactic acid andDL-malic acid were examined. 25 ml of each culture medium was placed ina 250-ml Erlenmeyer flask and incubated at 32° C. with shaking. Theamount of L-pipecolic acid accumulated after 24 hours of incubation was4.8 g/l, 3.3 g/l, 2.8 g/l, 2.6 g/l, 3.5 g/l, 4.7 g/l and 4.7 g/l,respectively. This indicates that organic acids can be used as carbonsources in the present invention.

Example 4 (Production of L-Pipecolic Acid With Disintegrated BacterialCells)

[0071] Each of E. coli RK4904pUC19 strain, E. coli RK4904pUClat strain,E. coli RK4904pUCproC strain and E. coli RK4904pUClatproC strain wasinoculated into 3 ml of L medium containing 50 μg/ml ampicillin sodium,and incubated at 32° C. overnight with shaking. The resulting cultureswere used seed cultures, and 275 μl of each seed culture was inoculatedinto 50 ml of L medium containing 50 μg/ml ampicillin sodium, andincubated at 32° C. for 8 hours with shaking. After this culture wascentrifuged, the cells were washed with 0.85% NaCl and 2 ml of BugBuster(Novagen) was added thereto. The resulting supernatant was used as adisintegrated cell suspension. To 100 μl of each disintegrated cellsuspension was added 1 ml of a 0.2 M phosphate buffer (pH 7.2)containing 20 μmol L-lysine-HCl, 20 μmol 2-ketoglutaric acid, 0.075 μmolpyridoxal phosphate and 200 μmol β-NAD⁺, followed by standing at 32° C.for 15 hours. 5 μl each of the resulting reaction mixtures were spottedonto a TLC plate (Merck Art. 13143), developed with a developing solvent(1-butanol-acetic acid-water=3:1:1), and then treated with ninhydrin toproduce a color. As a result, a spot of L-pipecolic acid was observedfor the disintegrated cell suspension of E. coli RK4904pUClatproCstrain. This indicates that, also in an in vitro reaction, L-pipecolicacid is produced by the action of LAT formed from the lat gene encodedon the plasmid and pyrroline-5-carboxylate reductase formed from proC.

Example 5

[0072] Similarly to pUClatlysPLargT-tet, the plasmid pUClatlysPL-tet wasconstructed by introducing the tetracycline resistance gene into theScaI site of pUClatlysPL. Using these plasmids, the following conversionreaction was carried out.

[0073] Each of the frozen seed cultures of two bacterial strains, E.coli BL21 pUClatlysPLargT-tet strain and E. coli BL21 pUClatlysPL-tetstrain, was inoculated into L medium (3 ml/centrifuge tube) containing25 μg/ml tetracycline, and incubated at 32° C. for 18 hours on a rotaryshaker. Then, 500 μl of each seed cultures was inoculated into TB medium(27.5 ml/centrifuge tube) containing 25 μg/ml tetracycline, andincubated at 32° C. for 24 hours on a rotary shaker. After completion ofthe incubation, the O.D. at 660 nm was 3.98 and 9.38, respectively.Cells were collected from each of the resulting cultures (4.71 ml and2.00 ml, respectively). After 1 ml of a 25 mM phosphate buffer (pH 6.8)was added to the collected cells, 20 μl of a 20% L-lysine solution and20 μl of 20% glycerol were added to initiate the conversion reaction at32° C. Two hours, 7 hours and 24 hours after the start of the reaction,100-μl samples were taken and their pipecolic acid and lysine contentswere determined by HPLC.

[0074] The amounts of pipecolic acid accumulated after 2 hours, 7 hoursand 24 hours of the conversion reaction were 0.36 g/l, 0.73 g/l and 2.0g/l, respectively, for E. coli BL21 pUClatlysPLargT-tet strain; and 0.88g/l, 1.3 g/l and 1.4 g/l, respectively, for E. coli BL21 pUClatlysPL-tetstrain. Thus, the pipecolic acid production rate of E. coli BL21pUClatlysPL-tet strain decreased gradually, whereas E. coli BL21pUClatlysPLargT-tet strain maintained an almost constant pipecolic acidproduction rate, though its initial pipecolic acid production rate wassomewhat inferior. This pipecolic acid production rate correspondedexactly with the lysine consumption rate. The role of the argT gene inpipecolic acid production was confirmed by these experimental results.

Example 6

[0075] A culture test was carried out with respect to the lattransformed strain of a coryneform bacterium, namely C. glutamiciumATCC31831 pClat strain. As a control, C. glutamicum ATCC31831 pC2 strainhaving a lat-free plasmid was also tested in the same manner. Afterthese two strains were grown in agar plates to form colonies, thesecolonies were inoculated into test tubes in which 3 ml of L mediumcontaining 20 μg/ml kanamycin was placed, and incubated at 32° C. for 18hours on a rotary shaker. 275 μl each of the resulting cultures wereinoculated into 250-ml Erlenmeyer flasks in which 27.5 ml of TB mediumcontaining 20 μg/ml kanamycin was placed, and incubated at 32° C. on arotary shaker. Five hours, 15 hours, 39 hours and 63 hours after thestart of the incubation, 550 μl portions of 50% glycerol dissolved in aphosphate buffer (pH 6.8) were added thereto. The incubation wasdiscontinued 135 hours after the start of the addition of glycerol, andthe pipecolic acid content of the culture supernatant was determined byHPLC. As a result, C. glutamicum ATCC31831 pClat strain accumulatedabout 0.7 g/l of pipecolic acid in 135 hours after the first addition ofglycerol. On the other hand, the control (C. glutamicum ATCC31831 pC2strain) showed no accumulation of pipecolic acid. This indicates thatthe lat gene introduced by means of a plasmid is also expressed in thecoryneform bacterium and that the pyrroline-5-carboxylate reductase(proC gene) of the coryneform bacterium reduces P6C converted fromlysine by the action of LAT and thereby produces pipecolic acid.

1 20 1 2663 DNA Flavobacterium lutescens 1 cccgggtgtc attgaataccagcaggtcgc caggttgcag cagctggtcc agatcgcgca 60 cctggcgatc ctccagcgcagccggtgccg gcggcaccag cagcaggcgg ctggccgaac 120 gctccggcag cggcgcctgggcaatcagtt cgggaggcag gtggtaggca aaatcggact 180 tcttcaacgc cggcagctcgatacaacggg ggcgtcagtt tacgcccctg taccgcctgt 240 gccctcaccg ctcgaacttggtgcccagga tcaccgccgt ggtggtgcgc tcgaccccat 300 cagtggcgcc gatggcatcggtcagctcgt ccatcgccgc cacgccatcg acggcggcca 360 tcgccaccag gtcatgcgcgccactgaccg aatgcaggct gcgcaccgca gcaatggcct 420 gcagcgcccg cacgaccgccggcattttct tcggcatcac ggtgatggag atatgcgcgc 480 ggacctgctg gcgctccatcgcctggccaa ggcgcacggt gtagccggcg attattccgc 540 tgtgctgcag ccgctcgatccggctctgca ccgtggtccg cgacaccccg agccggcgcg 600 ccagcgccgc ggtcgaggcgcgcgcatcct cacgcaacag gtcaagcaac tgtgcatccg 660 cctgggaaat ggtcactttgtcgaaaacct ttcgtcaatc cgccgaaacc ggccattgat 720 ttgagcagat tcgcactgccatttgtagtg cgttaacggt tacaactaac actagacaca 780 atcagcacgg attcagc atgtcc ctt ctt gcc ccg ctc gcc ccg ctc cgc 830 Met Ser Leu Leu Ala Pro LeuAla Pro Leu Arg 1 5 10 gcc cat gcc ggc acc cgc ctt acc cag ggc ctg tctgac ccg cag gtc 878 Ala His Ala Gly Thr Arg Leu Thr Gln Gly Leu Ser AspPro Gln Val 15 20 25 gag cag ctg gcc gcc aac cac cct gac ctg cgc gcc gccatc gac gcc 926 Glu Gln Leu Ala Ala Asn His Pro Asp Leu Arg Ala Ala IleAsp Ala 30 35 40 gct gcc gac gaa tac gcg cgc atc aaa ccg cag gcc gcg gcattg ctg 974 Ala Ala Asp Glu Tyr Ala Arg Ile Lys Pro Gln Ala Ala Ala LeuLeu 45 50 55 gac ctg gat gaa agc gcg cag atc gcc gcc gtg cag gat ggc ttcgtc 1022 Asp Leu Asp Glu Ser Ala Gln Ile Ala Ala Val Gln Asp Gly Phe Val60 65 70 75 aac ttc tat gcc gat gat gcg gtg gtg ccc tat atc gcc ctg gccgcc 1070 Asn Phe Tyr Ala Asp Asp Ala Val Val Pro Tyr Ile Ala Leu Ala Ala80 85 90 cgc ggg ccg tgg gtg gtc agc ctg aag ggc gcg gtg ctg tat gac gcc1118 Arg Gly Pro Trp Val Val Ser Leu Lys Gly Ala Val Leu Tyr Asp Ala 95100 105 ggc ggc tac ggc atg ctc ggc ttc ggc cat acc ccg gcc gat atc ctg1166 Gly Gly Tyr Gly Met Leu Gly Phe Gly His Thr Pro Ala Asp Ile Leu 110115 120 gag gcg gtc ggc aag ccg cag gtg atg gcc aac atc atg act ccc tcg1214 Glu Ala Val Gly Lys Pro Gln Val Met Ala Asn Ile Met Thr Pro Ser 125130 135 ctg gcc cag ggc cgc ttc att gcc gca atg cgc cgc gaa atc ggc cat1262 Leu Ala Gln Gly Arg Phe Ile Ala Ala Met Arg Arg Glu Ile Gly His 140145 150 155 acc cgc ggc ggc tgc ccg ttc tcg cac ttc atg tgc ctg aac tccggc 1310 Thr Arg Gly Gly Cys Pro Phe Ser His Phe Met Cys Leu Asn Ser Gly160 165 170 tcc gaa gcg gtc ggg ctg gcc gcg cgc atc gcc gac atc aac gccaag 1358 Ser Glu Ala Val Gly Leu Ala Ala Arg Ile Ala Asp Ile Asn Ala Lys175 180 185 ctg atg acc gac ccg ggc gcc cgg cat gcc ggc gcc acg atc aagcgc 1406 Leu Met Thr Asp Pro Gly Ala Arg His Ala Gly Ala Thr Ile Lys Arg190 195 200 gtg gtg atc aag ggc agt ttc cac ggc cgt acc gac cgt ccg gcgctg 1454 Val Val Ile Lys Gly Ser Phe His Gly Arg Thr Asp Arg Pro Ala Leu205 210 215 tat tcc gat tcc acc cgc aag gcc tac gat gcg cat ctg gcc agctac 1502 Tyr Ser Asp Ser Thr Arg Lys Ala Tyr Asp Ala His Leu Ala Ser Tyr220 225 230 235 cgc gac gag cac agc gtc att gcc atc gcc ccg tat gac cagcag gcc 1550 Arg Asp Glu His Ser Val Ile Ala Ile Ala Pro Tyr Asp Gln GlnAla 240 245 250 ctg cgc cag gtg ttt gcc gat gcc cag gcc aac cac tgg ttcatc gag 1598 Leu Arg Gln Val Phe Ala Asp Ala Gln Ala Asn His Trp Phe IleGlu 255 260 265 gcg gtg ttc ctg gag ccg gtg atg ggc gaa ggc gac ccg ggccgt gcg 1646 Ala Val Phe Leu Glu Pro Val Met Gly Glu Gly Asp Pro Gly ArgAla 270 275 280 gtg ccg gtg gac ttc tac cgc ctg gcc cgt gag ctg acc cgcgaa cac 1694 Val Pro Val Asp Phe Tyr Arg Leu Ala Arg Glu Leu Thr Arg GluHis 285 290 295 ggc agc ctg ctg ctg atc gat tcg atc cag gcc gcg ctg cgcgtg cac 1742 Gly Ser Leu Leu Leu Ile Asp Ser Ile Gln Ala Ala Leu Arg ValHis 300 305 310 315 ggc acc ctg tcc ttc gtc gac tac ccc ggc cac cag gagctg gag gca 1790 Gly Thr Leu Ser Phe Val Asp Tyr Pro Gly His Gln Glu LeuGlu Ala 320 325 330 ccg gac atg gag acc tac tcc aag gcc ctg aac ggc gcccag ttc ccg 1838 Pro Asp Met Glu Thr Tyr Ser Lys Ala Leu Asn Gly Ala GlnPhe Pro 335 340 345 ctg tcg gta gtg gcc gtg acc gag cac gcc gcc gcg ctgtac cgc aag 1886 Leu Ser Val Val Ala Val Thr Glu His Ala Ala Ala Leu TyrArg Lys 350 355 360 ggc gtg tac ggc aac acc atg acc acc aac ccg cgg gcgctg gac gtg 1934 Gly Val Tyr Gly Asn Thr Met Thr Thr Asn Pro Arg Ala LeuAsp Val 365 370 375 gcc tgc gcc acc ctg gca cgc ctg gat gag ccg gtc cgcaac aat atc 1982 Ala Cys Ala Thr Leu Ala Arg Leu Asp Glu Pro Val Arg AsnAsn Ile 380 385 390 395 cgc ctg cgt ggc cag cag gcg atg cag aag ctg gaagca ttg aag gaa 2030 Arg Leu Arg Gly Gln Gln Ala Met Gln Lys Leu Glu AlaLeu Lys Glu 400 405 410 cgg ctg ggg ggc gcg atc acc aag gtg cag ggc accggc ctg ctg ttc 2078 Arg Leu Gly Gly Ala Ile Thr Lys Val Gln Gly Thr GlyLeu Leu Phe 415 420 425 tcc tgc gag ctg gcc ccg cag tac aag tgc tac ggggcc ggc tcc acc 2126 Ser Cys Glu Leu Ala Pro Gln Tyr Lys Cys Tyr Gly AlaGly Ser Thr 430 435 440 gag gag tgg ctg cgc atg cac ggg gtc aat gtg atccac ggc ggc gag 2174 Glu Glu Trp Leu Arg Met His Gly Val Asn Val Ile HisGly Gly Glu 445 450 455 aat tcg ctg cgc ttc acc ccg cac ttc ggc atg gacgag gcc gaa ctg 2222 Asn Ser Leu Arg Phe Thr Pro His Phe Gly Met Asp GluAla Glu Leu 460 465 470 475 gac ctg ctg gtg gag atg gtc ggg cgt gcg ctggtc gaa ggc cca cgc 2270 Asp Leu Leu Val Glu Met Val Gly Arg Ala Leu ValGlu Gly Pro Arg 480 485 490 cgg gcc tga tccgcacccg caggacggaa ggcacgagcccaccgtgagg cgggctctt 2328 Arg Ala tgctgcccgg caccagcggc aacaggccgcgctgtcaccg gccaggcggg gcgccggcag 2388 tgggtttcag ccgcaggggt ccgccctgccagcgcctgcg gcggggcaca ggcttgcggg 2448 cattgcggcc tctgccacgg gcacgcagccggagatcagg ctgacaaggg ggctgccccg 2508 ggtggcagta cacgaccagc cagttgactgccggtatttg cttgatcagc gctgcatcca 2568 gaacagcacc atcggttgcg tgactgacgcgccgctggcc gttgcgggac agcagccttt 2628 gcgtcacacg tggcccgcac ctgcctgcactgcag 2663 2 30 DNA Artificial Sequence Synthesized to alter a region inthe neighborhood of the N-terminal ATG of the gene of Flavobacteriumlutescens encoding lysine 6-aminotransferase (the lat gene) to a NdeIsite 2 tccatatgtc ccttcttgcc ccgctcgccc 30 3 30 DNA Artificial SequenceSynthesized to alter a region downstream of the termination codon of thegene of Flavobacterium lutescens encoding lysine 6-aminotransferase (thelat gene) to a BamHI site 3 gcggatcctg ttgccgctgg tgccgggcag 30 4 29 DNAArtificial Sequence Synthesized to alter a region in the neighborhood ofthe N-terminal ATG of the gene of Flavobacterium lutescens encodinglysine 6-aminotransferase (the lat gene) to a HindIII site 4 ataagcttgtcccttcttgc cccgctcgc 29 5 1837 DNA Escherichia coli 5 atttagccacgactacgttg cacttccagc caccacttct ccagctccgc ggcaaaggcc 60 tggcggtcacgttgtgaaag gctatctggt ccgccggtct ggatcccact ggcacgtaag 120 gtatccataaaatcgcgcat cgtcaggcgc tcacgaatgg tcgctggcgt gtaacgttcg 180 ccgcgcggattaagcgccgc agcgcctttc tcgatggctt cagcagccaa aggtatatct 240 gcggtgatcaccaaatcgcc cgcttcacac tgccggacaa tttcgttatc ggcaacgtcg 300 aaacctgccgcgacgcgcag cgtacgaata aatcgcgatg gcggcacgcg taaactctgg 360 tttgctaccagtaccagcgg catctgcata cgttccgccg cgcgatacaa aatctcttta 420 attacattgggacacgcgtc ggcatccacc caaattgtca taaagtcatc ctttgttggg 480 taatcctctattgtgtcgcg cttttgcctt ccggcatagt tctgtttatg cttctgccag 540 cgattatcaaaacaatgaat ttcacggcag gagtgaggca atg gaa aag aaa atc 595 Met Glu Lys LysIle 1 5 ggt ttt att ggc tgc ggc aat atg gga aaa gcc att ctc ggc ggt ctg643 Gly Phe Ile Gly Cys Gly Asn Met Gly Lys Ala Ile Leu Gly Gly Leu 1015 20 att gcc agc ggt cag gtg ctt cca ggg caa atc tgg gta tac acc ccc691 Ile Ala Ser Gly Gln Val Leu Pro Gly Gln Ile Trp Val Tyr Thr Pro 2530 35 tcc ccg gat aaa gtc gcc gcc ctg cat gac cag ttc ggc atc aac gcc739 Ser Pro Asp Lys Val Ala Ala Leu His Asp Gln Phe Gly Ile Asn Ala 4045 50 gca gaa tcg gcg caa gaa gtg gcg caa atc gcc gac atc att ttt gct787 Ala Glu Ser Ala Gln Glu Val Ala Gln Ile Ala Asp Ile Ile Phe Ala 5560 65 gcc gtt aaa cct ggc atc atg att aaa gtg ctt agc gaa atc acc tcc835 Ala Val Lys Pro Gly Ile Met Ile Lys Val Leu Ser Glu Ile Thr Ser 7075 80 85 agc ctg aat aaa gac tct ctg gtc gtt tct att gct gca ggt gtc acg883 Ser Leu Asn Lys Asp Ser Leu Val Val Ser Ile Ala Ala Gly Val Thr 9095 100 ctc gac cag ctt gcc cgc gcg ctg ggc cat gac cgg aaa att atc cgc931 Leu Asp Gln Leu Ala Arg Ala Leu Gly His Asp Arg Lys Ile Ile Arg 105110 115 gcc atg ccg aac act ccc gca ctg gtt aat gcc ggg atg acc tcc gta979 Ala Met Pro Asn Thr Pro Ala Leu Val Asn Ala Gly Met Thr Ser Val 120125 130 acg cca aac gcg ctg gta acc cca gaa gat acc gct gat gtg ctg aat1027 Thr Pro Asn Ala Leu Val Thr Pro Glu Asp Thr Ala Asp Val Leu Asn 135140 145 att ttc cgc tgc ttt ggc gaa gcg gaa gta att gct gag ccg atg atc1075 Ile Phe Arg Cys Phe Gly Glu Ala Glu Val Ile Ala Glu Pro Met Ile 150155 160 165 cac ccg gtg gtc ggt gtg agc ggt tct tcg cca gcc tac gta tttatg 1123 His Pro Val Val Gly Val Ser Gly Ser Ser Pro Ala Tyr Val Phe Met170 175 180 ttt atc gaa gcg atg gcc gac gcc gcc gtg ctg ggc ggg atg ccacgc 1171 Phe Ile Glu Ala Met Ala Asp Ala Ala Val Leu Gly Gly Met Pro Arg185 190 195 gcc cag gcg tat aaa ttt gcc gct cag gcg gta atg ggt tcc gcaaaa 1219 Ala Gln Ala Tyr Lys Phe Ala Ala Gln Ala Val Met Gly Ser Ala Lys200 205 210 atg gtg ctg gaa acg gga gaa cat ccg ggg gca ctg aaa gat atggtc 1267 Met Val Leu Glu Thr Gly Glu His Pro Gly Ala Leu Lys Asp Met Val215 220 225 tgc tca ccg gga ggc acc acc att gaa gcg gta cgc gta ctg gaagag 1315 Cys Ser Pro Gly Gly Thr Thr Ile Glu Ala Val Arg Val Leu Glu Glu230 235 240 245 aaa ggc ttc cgt gct gca gtg atc gaa gcg atg acg aag tgtatg gaa 1363 Lys Gly Phe Arg Ala Ala Val Ile Glu Ala Met Thr Lys Cys MetGlu 250 255 260 aaa tca gaa aaa ctc agc aaa tcc tga tgactttcgccggacgtcag gccgcca 1417 Lys Ser Glu Lys Leu Ser Lys Ser 265 cttcggtgcggttacgtccg gctttctttg ctttgtaaag cgccaaatct gccgatttca 1477 accactcacgatagtgactc atttgtgggt tcagcggcgc aacccccaca ctaatccgta 1537 aagttacctgtggcgtattc ggcaaacgta atgtatttag cccttcatgc acccgtaaca 1597 tggcggtaatggcgctctca gctggcgtac cggacatgat tactgcaaac tcatcgccgc 1657 caaaccgaccaatcacatcg ctaccgcgca gggtaatttg taactgtcgg gtaagcgcca 1717 caatcgcttcatcgcccaca tcatggcccc aggtatcgtt gatgctcttg aaatggtcga 1777 tatcgataatcagtaacgtt gcatcgcgat tatgccgccg acagttatca aattcattgc 1837 6 30 DNAArtificial Sequence Synthesized to alter a region in the neighborhood ofthe N-terminal ATG of the gene of Escherichia coli encodingpyrroline-5-carboxylate reductase (the proC gene) to a KpnI site 6agggtaccat aaaatcgcgc atcgtcaggc 30 7 30 DNA Artificial SequenceSynthesized to alter a region downstream of the termination codon of theproC gene of Escherichia coli to a KpnI site 7 ccggtaccgc cacaggtaactttacggatt 30 8 2186 DNA Escherichia coli 8 ggatcctgcg tgaacgcggttccggcacgc gggagattgt cgattatctg ttgctgtcac 60 atttaccgaa gtttgagatggcgatggaat taggtaactc cgaggcaatc aaacatgcgg 120 tgcgtcatgg gttgggaattagttgcctgt cgcgacgtgt gattgaagat caattgcagg 180 caggcacatt aagtgaagttgcggtccctc tgccgcgcct gatgcgtacg ttgtggcgta 240 tacatcatcg gcaaaaacacctttccaacg cgctacggcg ctttctggac tattgcgatc 300 ccgcaaatgt gccgcgttaagttgctgtac aagaacatgc tggtgctgtg tcgatttcgt 360 gacgcagcgc cttcagcatgcattcgccag aaaagagatt ggctgcttta cttataatcc 420 ctgggcgatc atgaaggtgtcttataaccg tgtatttctg ccggaaggat tgccaatcgt 480 ctgctacaat cgcgcctcatttttaagatg gatagcattt ttgt atg gtt tcc gaa 536 Met Val Ser Glu 1 act aaaacc aca gaa gcg ccg ggc tta cgc cgt gaa tta aag gcg cgt 584 Thr Lys ThrThr Glu Ala Pro Gly Leu Arg Arg Glu Leu Lys Ala Arg 5 10 15 20 cac ctgacg atg att gcc att ggc ggt tcc atc ggt aca ggt ctt ttt 632 His Leu ThrMet Ile Ala Ile Gly Gly Ser Ile Gly Thr Gly Leu Phe 25 30 35 gtt gcc tctggc gca acg att tct cag gca ggt ccg ggc ggg gca ttg 680 Val Ala Ser GlyAla Thr Ile Ser Gln Ala Gly Pro Gly Gly Ala Leu 40 45 50 ctc tcg tat atgctg att ggc ctg atg gtt tac ttc ctg atg acc agt 728 Leu Ser Tyr Met LeuIle Gly Leu Met Val Tyr Phe Leu Met Thr Ser 55 60 65 ctc ggt gaa ctg gctgca tat atg ccg gtt tcc ggt tcg ttt gcc act 776 Leu Gly Glu Leu Ala AlaTyr Met Pro Val Ser Gly Ser Phe Ala Thr 70 75 80 tac ggt cag aac tat gttgaa gaa ggc ttt ggc ttc gcg ctg ggc tgg 824 Tyr Gly Gln Asn Tyr Val GluGlu Gly Phe Gly Phe Ala Leu Gly Trp 85 90 95 100 aac tac tgg tac aac tgggcg gtg act atc gcc gtt gac ctg gtt gca 872 Asn Tyr Trp Tyr Asn Trp AlaVal Thr Ile Ala Val Asp Leu Val Ala 105 110 115 gct cag ctg gtc atg agctgg tgg ttc ccg gat aca ccg ggc tgg atc 920 Ala Gln Leu Val Met Ser TrpTrp Phe Pro Asp Thr Pro Gly Trp Ile 120 125 130 tgg agt gcg ttg ttc ctcggc gtt atc ttc ctg ctg aac tac atc tca 968 Trp Ser Ala Leu Phe Leu GlyVal Ile Phe Leu Leu Asn Tyr Ile Ser 135 140 145 gtt cgt ggc ttt ggt gaagcg gaa tac tgg ttc tca ctg atc aaa gtc 1016 Val Arg Gly Phe Gly Glu AlaGlu Tyr Trp Phe Ser Leu Ile Lys Val 150 155 160 acg aca gtt att gtc tttatc atc gtt ggc gtg ctg atg att atc ggt 1064 Thr Thr Val Ile Val Phe IleIle Val Gly Val Leu Met Ile Ile Gly 165 170 175 180 atc ttc aaa ggc gcgcag cct gcg ggc tgg agc aac tgg aca atc ggc 1112 Ile Phe Lys Gly Ala GlnPro Ala Gly Trp Ser Asn Trp Thr Ile Gly 185 190 195 gaa gcg ccg ttt gctggt ggt ttt gcg gcg atg atc ggc gta gct atg 1160 Glu Ala Pro Phe Ala GlyGly Phe Ala Ala Met Ile Gly Val Ala Met 200 205 210 att gtc ggc ttc tctttc cag gga acc gag ctg atc ggt att gct gca 1208 Ile Val Gly Phe Ser PheGln Gly Thr Glu Leu Ile Gly Ile Ala Ala 215 220 225 ggc gag tcc gaa gatccg gcg aaa aac att cca cgc gcg gta cgt cag 1256 Gly Glu Ser Glu Asp ProAla Lys Asn Ile Pro Arg Ala Val Arg Gln 230 235 240 gtg ttc tgg cga atcctg ttg ttc tat gtg ttc gcg atc ctg att atc 1304 Val Phe Trp Arg Ile LeuLeu Phe Tyr Val Phe Ala Ile Leu Ile Ile 245 250 255 260 agc ctg att attccg tac acc gat ccg agc ctg ctg cgt aac gat gtt 1352 Ser Leu Ile Ile ProTyr Thr Asp Pro Ser Leu Leu Arg Asn Asp Val 265 270 275 aaa gac atc agcgtt agt ccg ttc acc ctg gtg ttc cag cac gcg ggt 1400 Lys Asp Ile Ser ValSer Pro Phe Thr Leu Val Phe Gln His Ala Gly 280 285 290 ctg ctc tct gcggcg gcg gtg atg aac gca gtt att ctg acg gcg gtg 1448 Leu Leu Ser Ala AlaAla Val Met Asn Ala Val Ile Leu Thr Ala Val 295 300 305 ctg tca gcg ggtaac tcc ggt atg tat gcg tct act cgt atg ctg tac 1496 Leu Ser Ala Gly AsnSer Gly Met Tyr Ala Ser Thr Arg Met Leu Tyr 310 315 320 acc ctg gcg tgtgac ggt aaa gcg ccg cgc att ttc gct aaa ctg tcg 1544 Thr Leu Ala Cys AspGly Lys Ala Pro Arg Ile Phe Ala Lys Leu Ser 325 330 335 340 cgt ggt ggcgtg ccg cgt aat gcc ctg tat gcg acg acg gtg att gcc 1592 Arg Gly Gly ValPro Arg Asn Ala Leu Tyr Ala Thr Thr Val Ile Ala 345 350 355 ggt ctg tgcttc ctg acc tcc atg ttt ggc aac cag acg gta tac ctg 1640 Gly Leu Cys PheLeu Thr Ser Met Phe Gly Asn Gln Thr Val Tyr Leu 360 365 370 tgg ctg ctgaac acc tcc ggg atg acg ggt ttt atc gcc tgg ctg ggg 1688 Trp Leu Leu AsnThr Ser Gly Met Thr Gly Phe Ile Ala Trp Leu Gly 375 380 385 att gcc attagc cac tat cgc ttc cgt cgc ggt tac gta ttg cag gga 1736 Ile Ala Ile SerHis Tyr Arg Phe Arg Arg Gly Tyr Val Leu Gln Gly 390 395 400 cac gac attaac gat ctg ccg tac cgt tca ggt ttc ttc cca ctg ggg 1784 His Asp Ile AsnAsp Leu Pro Tyr Arg Ser Gly Phe Phe Pro Leu Gly 405 410 415 420 ccg atcttc gca ttc att ctg tgt ctg att atc act ttg ggc cag aac 1832 Pro Ile PheAla Phe Ile Leu Cys Leu Ile Ile Thr Leu Gly Gln Asn 425 430 435 tac gaagcg ttc ctg aaa gat act att gac tgg ggc ggc gta gcg gca 1880 Tyr Glu AlaPhe Leu Lys Asp Thr Ile Asp Trp Gly Gly Val Ala Ala 440 445 450 acg tatatt ggt atc ccg ctg ttc ctg att att tgg ttc ggc tac aag 1928 Thr Tyr IleGly Ile Pro Leu Phe Leu Ile Ile Trp Phe Gly Tyr Lys 455 460 465 ctg attaaa gga act cac ttc gta cgc tac agc gaa atg aag ttc ccg 1976 Leu Ile LysGly Thr His Phe Val Arg Tyr Ser Glu Met Lys Phe Pro 470 475 480 cag aacgat aag aaa taagtttcct cccttccttg ctaagccctc tcaaccgaga 2031 Gln Asn AspLys Lys 485 gggctttttc aattccattt ccctgacaaa tcatgcggat ataaaatttaacatttggat 2091 tgataattgt tatcgtttgc attatcgtta cgccgcaatc aaaaaaggctgacaaatcag 2151 aggctgttcc ggctttctgg gatggatcca gatct 2186 9 30 DNAArtificial Sequence Synthesized to alter a region in the neighborhood ofthe N-terminal ATG of the gene of Escherichia coli encoding alysine-specific permease (lysP) to a BglII site 9 tgagatctgg atcctgcgtgaacgcggttc 30 10 30 DNA Artificial Sequence Synthesized to alter aregion downstream of the termination codon of the lysP gene ofEscherichia coli to a BamHI site 10 gcagatctgg atcccagaaa gccggaacag 3011 2150 DNA Escherichia ?oli 11 tttgccagac cgccagcagc actaacgtgcgctccaatgt atttcatgcg aggactcctg 60 ttaaacccgc tggaggaaaa cggtaatgatagcgggttaa caggagcaag atgtagtggt 120 ttatgcgatg acgctttgaa tcacatagttaatcgcacca ccaccaacaa tcagccaggc 180 aaacagtacc agtgccatca gcagaggtttcgccccagct tttttcagcg cgctgacgtg 240 agtggtcaga cccagcgccg ccatcgccattgccagcagg aaggtatcca gcgttaccag 300 catgttcacc acgctctgcg gtaacaggtggaacgagtta aagatggcaa ctacgatgaa 360 caagatggca aaccacggaa tagtgattttgcttttctcg ccgctgttcg ccccagacag 420 ctgtttaaca cgcgccgcca gcaggatgaggaacggagcc agcatcatca cgcgcagcat 480 tttggaaata actgctgcgt tttccgcatccgggctgatg gcatgacctg ccgccaccac 540 ctgcgccact tcgtgcacag tagaaccaatgtagataccg aaagtttccg gactaaacca 600 ttgagacatc agcggatata tcgccgggtagaggaaaatc gcgacggtcc cgaagataac 660 aacggttgca acagccacgg ttactttactggcttccgct ttcactaccg gctcagtcgc 720 cagtaccgcg gcagcaccac agatactgctaccggcaccg atcaaccagc tggtgtgctt 780 atccagacca aacactttct gccccaggaagcaagccagc aggaaggtac tggacagcgt 840 caacacgtca atgatgatcc cactgataccgacatcggca atttgcgaga acgtcagacg 900 gaagccataa agaatgatac ccagacgtaataaatattgc ttggcaaaca gcacaccacc 960 gtcacagctt ttccagatgt gcggatagatggtgttgcct aaaaccatcc ccaacaagat 1020 tgcgagggtg agggcactaa acccggcacccgcaaccgcg ggaatggaac caccccacag 1080 ggcgaccccg gtgataactg cactcagggctaaccccgga ataaaatgcc acagtgtacg 1140 atgttgtttc tgtaaggtga tattcgtcataaccctctcc tttaaccggg cataaggtta 1200 cgactaactg gtttaaaaat aaaattgattatatatttat aattaatctt tataagtggt 1260 aagcgact atg cac atc acc ctc cggcag ttg gaa gtt ttt gca gaa gta 1310 Met His Ile Thr Leu Arg Gln Leu GluVal Phe Ala Glu Val 1 5 10 ttg aaa agt gga tca acc acc cag gcg tcg gtgatg ctg gcg ttg tcg 1358 Leu Lys Ser Gly Ser Thr Thr Gln Ala Ser Val MetLeu Ala Leu Ser 15 20 25 30 caa tca gca gtg agc gca gcc ttg acc gac ctggaa ggg cag ctt ggc 1406 Gln Ser Ala Val Ser Ala Ala Leu Thr Asp Leu GluGly Gln Leu Gly 35 40 45 gtg caa ctg ttt gat cgc gtg ggg aaa aga ctg gttgtt aat gaa cac 1454 Val Gln Leu Phe Asp Arg Val Gly Lys Arg Leu Val ValAsn Glu His 50 55 60 ggg cgg ctg ctc tat ccg cgt gcg ttg gca ttg ctt gaacag gcg gtt 1502 Gly Arg Leu Leu Tyr Pro Arg Ala Leu Ala Leu Leu Glu GlnAla Val 65 70 75 gaa atc gaa caa ctg ttt cgc gaa gac aac ggc gcg att cgtatc tat 1550 Glu Ile Glu Gln Leu Phe Arg Glu Asp Asn Gly Ala Ile Arg IleTyr 80 85 90 gcc agt agt acc atc ggt aac tac att ctg cct gca gtt atc gcccgt 1598 Ala Ser Ser Thr Ile Gly Asn Tyr Ile Leu Pro Ala Val Ile Ala Arg95 100 105 110 tat cgc cat gat tat ccg cag ttg ccg att gaa ctt agc gttggg aat 1646 Tyr Arg His Asp Tyr Pro Gln Leu Pro Ile Glu Leu Ser Val GlyAsn 115 120 125 agc cag gac gtg atg caa gcg gtg ctg gat ttc cgc gtt gatatt ggc 1694 Ser Gln Asp Val Met Gln Ala Val Leu Asp Phe Arg Val Asp IleGly 130 135 140 ttt att gaa gga ccg tgc cac agc act gaa atc att tct gaaccg tgg 1742 Phe Ile Glu Gly Pro Cys His Ser Thr Glu Ile Ile Ser Glu ProTrp 145 150 155 ctg gaa gac gag ctg gtg gtt ttc gcc gcg ccg act tcg ccgttg gcc 1790 Leu Glu Asp Glu Leu Val Val Phe Ala Ala Pro Thr Ser Pro LeuAla 160 165 170 cgt ggt ccg gtc acc tta gaa cag ctg gcc gct gcg ccg tggatc ctg 1838 Arg Gly Pro Val Thr Leu Glu Gln Leu Ala Ala Ala Pro Trp IleLeu 175 180 185 190 cgt gaa cgc ggt tcc ggc acg cgg gag att gtc gat tatctg ttg ctg 1886 Arg Glu Arg Gly Ser Gly Thr Arg Glu Ile Val Asp Tyr LeuLeu Leu 195 200 205 tca cat tta ccg aag ttt gag atg gcg atg gaa tta ggtaac tcc gag 1934 Ser His Leu Pro Lys Phe Glu Met Ala Met Glu Leu Gly AsnSer Glu 210 215 220 gca atc aaa cat gcg gtg cgt cat ggg ttg gga att agttgc ctg tcg 1982 Ala Ile Lys His Ala Val Arg His Gly Leu Gly Ile Ser CysLeu Ser 225 230 235 cga cgt gtg att gaa gat caa ttg cag gca ggc aca ttaagt gaa gtt 2030 Arg Arg Val Ile Glu Asp Gln Leu Gln Ala Gly Thr Leu SerGlu Val 240 245 250 gcg gtc cct ctg ccg cgc ctg atg cgt acg ttg tgg cgtata cat cat 2078 Ala Val Pro Leu Pro Arg Leu Met Arg Thr Leu Trp Arg IleHis His 255 260 265 270 cgg caa aaa cac ctt tcc aac gcg cta cgg cgc tttctg gac tat tgc 2126 Arg Gln Lys His Leu Ser Asn Ala Leu Arg Arg Phe LeuAsp Tyr Cys 275 280 285 gat ccc gca aat gtg ccg cgt taa 2150 Asp Pro AlaAsn Val Pro Arg 290 12 32 DNA Artificial Sequence Synthesized to attacha BglII site to a position in the neighborhood of the 5′-terminus of thelysP type transcriptional regular yeiE of Escherichia coli 12 atagatctcttgttgcctaa aaccatcccc aa 32 13 31 DNA Artificial Sequence Synthesized toattach a KpnI site to a position downstream of the termination codon ofthe lysP gene of Escherichia coli 13 gtggtacccc ccagaaagcc ggaacagcct c31 14 27 DNA Artificial Sequence Synthesized 14 tcggtacctc gacattttgtttctgcc 27 15 27 DNA Artificial Sequence Synthesized 15 atggtaccataaaattgacc atcaagg 27 16 30 DNA Artificial Sequence Synthesized 16ttagtactct tatcatcgat aagctttaat 30 17 30 DNA Artificial SequenceSynthesized 17 gcagtactac agttctccgc aagaattgat 30 18 1295 DNAEscherichia coli 18 cgatcaaatc ctcgacattt tgtttctgcc attcaatcgaaacgctgcga ttcaaccgct 60 atacctgcta tcttcaactt caggacaata atgcaacgtcttattaacat atttaacgtt 120 gaatgttact gttgtcgtca agatggcata agacctgcatgaaagagcct gcaaacacac 180 aacacaatac acaacataaa aaagccattt tcacttgagggttatgt atg aag aag 236 Met Lys Lys 1 tcg att ctc gct ctg tct ttg ttagtc ggt ctc tcc aca gcg gct tcc 284 Ser Ile Leu Ala Leu Ser Leu Leu ValGly Leu Ser Thr Ala Ala Ser 5 10 15 agc tat gcg gcg cta ccg gag acg gtacgt atc gga acc gat acc acc 332 Ser Tyr Ala Ala Leu Pro Glu Thr Val ArgIle Gly Thr Asp Thr Thr 20 25 30 35 tac gca ccg ttc tca tcg aaa gat gctaaa ggt gat ttt gtt ggc ttt 380 Tyr Ala Pro Phe Ser Ser Lys Asp Ala LysGly Asp Phe Val Gly Phe 40 45 50 gat atc gat ctc ggt aac gag atg tgc aaacgg atg cag gtg aaa tgt 428 Asp Ile Asp Leu Gly Asn Glu Met Cys Lys ArgMet Gln Val Lys Cys 55 60 65 acc tgg gtt gcc agt gac ttt gac gcg ctg atcccc tca ctg aaa gcg 476 Thr Trp Val Ala Ser Asp Phe Asp Ala Leu Ile ProSer Leu Lys Ala 70 75 80 aaa aaa atc gac gct att att tcg tcg ctt tcc attacc gat aaa cgt 524 Lys Lys Ile Asp Ala Ile Ile Ser Ser Leu Ser Ile ThrAsp Lys Arg 85 90 95 cag cag gag att gcc ttc tcc gac aag ctg tac gcc gcagat tct cgt 572 Gln Gln Glu Ile Ala Phe Ser Asp Lys Leu Tyr Ala Ala AspSer Arg 100 105 110 115 ttg att gcg gcc aaa ggt tca ccg att cag cca acgctg gat tca ctg 620 Leu Ile Ala Ala Lys Gly Ser Pro Ile Gln Pro Thr LeuAsp Ser Leu 120 125 130 aaa ggt aaa cat gtt ggt gtg ctg cag gga tca acccag gaa gct tac 668 Lys Gly Lys His Val Gly Val Leu Gln Gly Ser Thr GlnGlu Ala Tyr 135 140 145 gct aac gag acc tgg cgt agt aaa ggc gtg gat gtggtg gcc tat gcc 716 Ala Asn Glu Thr Trp Arg Ser Lys Gly Val Asp Val ValAla Tyr Ala 150 155 160 aac cag gat ttg gtc tat tcc gat ctg gct gca ggacgt ctg gat gct 764 Asn Gln Asp Leu Val Tyr Ser Asp Leu Ala Ala Gly ArgLeu Asp Ala 165 170 175 gcg tta caa gat gaa gtt gct gcc agc gaa gga ttcctc aag caa cct 812 Ala Leu Gln Asp Glu Val Ala Ala Ser Glu Gly Phe LeuLys Gln Pro 180 185 190 195 gct ggt aaa gat ttc gcc ttt gct ggc tca tcagta aaa gac aaa aaa 860 Ala Gly Lys Asp Phe Ala Phe Ala Gly Ser Ser ValLys Asp Lys Lys 200 205 210 tac ttc ggt gat ggc acc ggt gta ggg cta cgtaaa gat gat gct gaa 908 Tyr Phe Gly Asp Gly Thr Gly Val Gly Leu Arg LysAsp Asp Ala Glu 215 220 225 ctg acg gct gcc ttc aat aag gcg ctt ggc gagctg cgt cag gac ggc 956 Leu Thr Ala Ala Phe Asn Lys Ala Leu Gly Glu LeuArg Gln Asp Gly 230 235 240 acc tac gac aag atg gcg aaa aag tat ttc gacttt aat gtc tac ggt 1004 Thr Tyr Asp Lys Met Ala Lys Lys Tyr Phe Asp PheAsn Val Tyr Gly 245 250 255 gac tgatacgtcg ctgggaagct gtacctgatggaatgatcat catggtgc 1055 Asp 260 acgccaggtt tgttgcacta tcgtggtgcattgaaatgca tacttaagca tttttaatga 1115 aaaataatac gtctaacggg gcgggatattttgccttgat ggtcaatttt atggcacgat 1175 aagtgtaaca aacctgtaaa tattccctataaaaagactg tcagttgagg acattatgaa 1235 aaaactggtg ctatcgctct ctctggttctggccttctcc agcgcaactg cggcgtttgc 1295 19 29 DNA Artificial SequenceSynthesized 19 ggggtaccca tgtcccttct tgccccgct 29 20 28 DNA ArtificialSequence Synthesized 20 ggggatcccg cggcctgttg ccgctggt 28

1. A process for the production of L-pipecolic acid which comprises thestep of reducing delta-1-piperideine-6-carboxylic acid by the use ofpyrroline-5-carboxylate reductase.
 2. A process as claimed in claim 1wherein the pyrroline-5-carboxylate reductase is derived fromEscherichia coli or a coryneform bacterium.
 3. A process as claimed inclaim 1 or 2 wherein the delta-1-piperideine-6-carboxylic acid isobtained by the step of converting L-lysine by the use of lysine6-aminotransferase.
 4. A process as claimed in claim 3 wherein thelysine 6-aminotransferase is derived from Flavohacterium lutescens.
 5. Aprocess as claimed in claim 3 or 4 wherein the step of reducingdelta-1-piperideine-6-carboxylic acid and the step of convertingL-lysine into L-pipecolic acid by the use of lysine 6-aminotransferaseare carried out by use of an Escherichia coli strain or coryneformbacterium having pyrroline-5-carboxylate reductase and transformed witha gene encoding lysine 6-aminotransferase.
 6. A process as claimed inclaim 5 wherein the Escherichia coli strain or coryneform bacteriumcontains at least one gene selected from the group consisting of anexogenous gene encoding pyrroline-5-carboxylate reductase and anexogenous gene participating in the incorporation of lysine.
 7. Arecombinant strain of Escherichia coli or a coryneform bacterium whichcontains a gene encoding lysine 6-aminotransferase in expressible form.8. A recombinant strain of Escherichia coli as claimed in claim 7 whichfurther contains, in expressible form, at least one gene selected fromthe group consisting of an exogenous gene encodingpyrroline-5-carboxylate reductase and an exogenous gene participating inthe incorporation of lysine.
 9. A recombinant strain of Escherichia colias claimed in claim 8 wherein the gene participating in theincorporation of lysine is a gene encoding a lysine-specific permease.10. A recombinant strain of Escherichia coli or a coryneform bacteriumas claimed in claim 8 wherein the gene participating in theincorporation of lysine is a gene encoding a lysine-specific permeaseand further contains a sequence encoding a transcriptional regulationfactor arranged in tandem on the upstream side thereof.
 11. A processfor the production of L-pipecolic acid which comprises the steps ofculturing the recombinant strain of Escherichia coli strain orcoryneform bacterium of claim 7 or 10 in a L-lysine-containing medium,and harvesting L-pipecolic acid accumulated in the resulting culture.